Method for measuring pancreatic metabolism

ABSTRACT

Disclosed is a method for measuring pancreatic metabolism, which is characterized by the fact that the increase in  13 CO 2  in the exhaled breath is determined prior to and following the intravenous application of secretin as well as prior to and following the oral application of a  13 C triglyceride.

The present invention relates to a method for measuring the metabolismof the pancreas in exocrine pancreatic insufficiency (EPI) of slight,moderate and severe degrees.

Exocrine pancreatic insufficiency is characterized by a reduction ofenzyme release from the pancreas, especially amylase, lipase andchymotrypsin release, and the reduction of bicarbonate release.

The existing so-called “gold standard” of the determination of EPIcomprises intravenously infusing secretin over a period of two hours andin the second hour additionally caerulein while the duodenal secretionis aspirated and analyzed through a duodenal probe. This determinationmethod is tedious, expensive and very unpleasant to the patient andmoreover interference prone. In addition, many patients exhibit anallergic reaction to caerulein. In addition, the test is interferenceprone and therefore subject to a considerable error rate. When there isno standardized performing of the test, its evaluation depends on thereference values established in the respective laboratory (seeJ.-Matthias Löhr: Exokrine Pankreasinsuffizienz, 1st Edition, Bremen:Uni-Med, 2001).

A simple test, but which is not very exact either, consists in thedetermination of elastase in the stool.

A pancreatic functional test in which amylase is determined mainly hasbeen described in DE-C-44 26 204. In this test, natural corn starch isadministered and, after metabolization, secreted through the exhaled airas ¹³C-enriched carbon dioxide, and analyzed. The test is based on thefact that corn starch is naturally enriched with ¹³C atoms.

A disadvantage of this test is that only severe degree exocrinepancreatic insufficiency can be determined thereby. Inter alia, this isdue to the fact that the pancreas is not the only source of amylase andtherefore the test is interfered with by amylase in the saliva andgastric secretion, cf. Loser et al., Z. Gastroenterol. 1997; 35:187-194).

Further examinations of the functionality of the pancreas were developedwith ¹⁴C- and ¹³C-enriched substances, the ¹³C-substances beingpreferred because they do not load the patient with the radioactive ¹⁴Cisotope. As ¹³C-enriched test substances, cholesterol esters andtriglycerides were mainly studied, and in particular, the mixedtriglyceride glyceryl-1,3-dioctadecanoate-2-octanoate-1-¹³C (alsoreferred to as 1,3-distearyl-2-(carboxyl-¹³C)octanoylglycerol) wasstudied because pancreatic lipase first hydrolyzes the two stearylgroups, and only then the shorter ¹³C-octanoyl group is subjected tometabolism.

Comparisons of the studies with the “gold standard” and other methodshad the result that this method too is only suitable for diagnosingsevere and, to some extent, moderate degree pancreatic insufficiency. Inslight degree exocrine pancreatic insufficiency, this method fails (cf.Löser et al., Scand 3. Gastroenterol. 1998; 33: 327-334).

Thus, there is still a need for a simpler, and more convenient for thepatient, determination method for the functionality of the pancreas,especially in cases of slight and moderate degree exocrine pancreaticinsufficiency, by measuring the metabolism of the pancreas. The ratingis effected as follows:

Slight Pancreatic Insufficiency:

Reduction of bicarbonate by <50% or

Reduction of one enzyme by <50% or

Reduction of two enzymes by <25% or

Reduction of one enzyme and bicarbonate by <25%

Moderate Pancreatic Insufficiency:

Reduction of more than two enzymes and/or bicarbonate by 25 to 75%

Severe Pancreatic Insufficiency:

Reduction of all enzymes and/or bicarbonate secretion by >75%

(Lankisch et al., Dig. Dis. Sci. 1983, 28: 490-493)

All calculations are based on the lower limits (mean value minus twosigma) of a normal universe.

It has now been found that the measurement is enabled by the fact thatthe increase of ¹³CO₂ in the exhaled air is determined before and afterintravenous administration of secretin and before and after oraladministration of a ¹³C-mixed triglyceride.

For determining the increase of ¹³CO₂ in the exhaled air, the ratiobetween ¹³CO₂ and ¹²CO₂ is preferably measured. Preferably, IRMS isemployed for the measurement.

IRMS (isotope ratio mass spectrometry) has in the meantime become themost sensitive and best method for determining ¹³CO₂ in the exhaled air.

An alternative possibility is measurement by means of NDIR(non-dispersive infrared spectroscopy).

While according to a method of the prior art, such as that described inthe above mentioned Löser et al. (1998) reference, only the ¹³C-enrichedmixed triglyceride is administered and then the proportion of ¹³CO₂ inthe respiratory air is measured, the method according to the inventionis characterized in that the measurement is effected under the influenceof previous secretin administration.

A reduced metabolization is indicated by a deficiency of pancreaticenzymes as seen by a delayed or reduced release of ¹³CO₂ as compared tohealthy subjects, thus allowing a reliable diagnose.

In the method according to the invention, a breath sample is takenbefore the beginning of the test, and then further samples are taken atintervals of, for example, 15, 20 or 30 minutes. Usually, themeasurement is effected for a period of from two to five hours,typically four hours.

Preferably, the administration of secretin is begun first, and then, the¹³C-enriched mixed triglyceride is administered during the secretinadministration or directly after the end thereof.

The exhaled air can be stored in sample containers and measured afterthe test has been completed. It is also possible to transport therespiratory air containers.

As the mixed triglyceride with ¹³C-labeling, those are preferablyemployed in which the acid in the 2-position bears the ¹³C-labels.Typically, in the triglyceride employed according to the invention, the1 and 3 positions bear typical fatty acids with 12 to 20 carbon atomswhich may also contain double bonds. In the 2 position of thetriglyceride, there is a shorter acid with 2 to 12 carbon atoms. Oneparticularly preferred mixed triglyceride isglyceryl-1,3-dioctadecanoate-2-octanoate-1-¹³C, a mixed triglyceridewhich is available with high enrichment, for example, fromSigma-Aldrich.

In contrast to the “gold standard” in which secretin and thensimultaneously caerulein must be infused in the course of two hours, themethod according to the invention administers only secretin, and withina short time, preferably from 5 to 25 minutes, typically 15 minutes. Theoral administration of the ¹³C-triglyceride is effected by means of astandardized test meal in the method according to the invention as well.Instead of the previously employed test meal consisting of 250 mg ofmixed triglyceride homogenized with 10 g of chocolate cream in a 60° C.hot water bath and application after cooling to a piece of toast with 15g of butter, this mixed triglyceride may preferably be administered moresimply by stirring only 200 mg of mixed triglyceride, for example, with15 g of corn starch, 3 g of cocoa powder and 15 g of butter in 20 ml ofwarm milk to obtain a homogeneous mixture, followed by spreading it on aslice of toast. The critical thing is not the amount of fat and oil, butthe fact that the triglyceride can be easily mixed homogeneously withthe other components and applied to a slice of toast. In principle, itis also possible to provide the mixed triglyceride in ready-packedportions with other stable and sufficiently fat-containing components,which only remains to be spread onto the slice of toast.

When the test results of the method according to the invention werecompared with test results of the “gold standard”, it has been found atfirst that although the correspondence is sufficient, it is still notfully satisfactory. Then, further studies have shown that thereproducibility of the method according to the invention is very highand the test results are thus much closer together than expected fromthe comparison with the “gold standard”. When this astonishing resultwas checked, it was established that the “gold standard” evidently wasnever examined for its reproducibility because of its tediousness andunacceptability by the patient, and therefore, the deviations from theresults of the method according to the invention are not to beattributed to the method according to the invention, but to errorsources in the “gold standard” (cf. Exokrine Pankreasinsuffizienz 2001,UniMed Verlag A G, Bremen, Löhr J.-M. and Schneider H. T.).

Thus, the method according to the invention is superior to the “goldstandard” in many respects: The placing of the duodenal probe, which isextraordinarily unpleasant for the patients, is dispensed with. Thetwo-hour infusion of secretin and caerulein is dispensed with. Instead,only intravenous administration is effected within about 15 minutes, forexample, and only of secretin. Since there are patients who can toleratesecretin, but not caerulein, there are substantially less studies whichare discontinued for intolerance.

The cost for the test is about one tenth of the cost for the “goldstandard”. For the first time, it becomes possible to determine alsoslight degrees of exocrine pancreatic insufficiency, and to subjectpatients with this diagnosis to therapy. Moderate degree exocrinepancreatic insufficiency is also often curable or at least capable ofstabilization. Severe degree pancreatic insufficiency generally can nolonger be cured, but only treated for its symptoms. Of particularimportance is the recognition in good time of the form of pancreaticinsufficiency of slight and moderate degrees in children, all the moreso since children can be cured better and more easily as compared toadults.

Thus, this simple method is also suitable as a follow-up of an enzymesubstitution therapy.

According to the invention, a method for the determination of thefunctionality of the pancreas, namely the mild and moderate forms ofexocrine pancreatic insufficiency, is provided.

EXAMPLE

¹³C Respiratory Test with Secretin Stimulation

Before the ¹³C-mixed triglyceride respiratory test is applied, thepatient should be fasted for 6 hours (preferably over night). The testis to be performed in a resting position (sitting or lying).

The test begins with taking the first zero value respiratory sample(“zero value”). Thus, the patient blows into the sample containerlabeled “zero value” by means of a straw. Then, the patient isadministered 1 U of secretin per kg of body weight intravenously over 15min.

Secretin can be obtained, for example, as Secrelux® from Goldham PharmaGmbH, Germany. One CU (clinical unit) corresponds to 1 U (unit).

The patient now ingests the prepared test meal within 10 minutes.

During the next four hours after the end of the test meal, respiratorysamples are taken at the times established in the protocol and in theway as described above into the sample containers, which are eachunambiguously labeled.

Sample Taking: 00 15 min 10 min 15 30 50 70 90 110 130 150 170 190 210230 min secretin i.v. test meal min min min min min min min min min minmin min

FIG. 1 shows the results of the ¹³CO₂ respiratory air values for ahealthy subject (x) and for a subject with slight (♦), moderate (▴) andsevere (□) degree EPI.

FIG. 2 shows the cumulative ¹³CO₂ respiratory air values for a healthysubject (x) for a subject with slight (♦), moderate (▴) and severe (□)degree EPI.

1. A method for measuring the metabolism of the pancreas, characterizedin that the increase of ¹³CO₂ in the exhaled air is determined beforeand after intravenous administration of secretin and before and afteroral administration of a ¹³C-triglyceride.
 2. The method according toclaim 1, characterized in that said ¹³C-triglyceride is the mixedtriglyceride glyceryl-1,3-dioctadecanoate-2-octanoate-1-¹³C.
 3. Themethod according to claim 1, characterized in that the determination of¹³CO₂ is effected by IRMS or NDIR.
 4. The method according to claim 1,characterized in that 1 U of secretin per kg of body weight isadministered intravenously within about 15 to 30 minutes.
 5. The methodaccording to claim 1, characterized in that 200 mg of the mixedtriglyceride is administered with a test meal.
 6. A test kit containing:a test meal of corn starch and cocoa powder; a ¹³C-mixed triglyceride; asample container; instructions for use relating to a method according toclaim
 1. 7. A test meal according to claim 1, characterized in that said¹³C-mixed triglyceride isglyceryl-1,3-dioctadecanoate-2-octanoate-1-¹³C.